Portal de Periódicos da CAPES

Pathogenic Anti-Desmoglein MAbs Show Variable ELISA Activity because of Preferential Binding of Mature versus Proprotein Isoforms of Desmoglein 3

2009; Elsevier BV; Volume: 129; Issue: 9 Linguagem: Inglês

10.1038/jid.2009.41

1523-1747

Preety Sharma, Eun Jung Choi, Keiko Kuroda, Takahisa Hachiya, Ken Ishii, Aimee Payne,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

antigen desmoglein 3 pemphigus vulgaris TO THE EDITOR The desmosomal cadherins desmoglein (DSG) 3 and DSG1 are targets of autoantibodies in the potentially fatal blistering disease, pemphigus vulgaris (PV) (Stanley and Amagai, 2006Stanley J.R. Amagai M. Pemphigus, bullous impetigo, and the staphylococcal scalded-skin syndrome.New Engl J Med. 2006; 355: 1800-1810Crossref PubMed Scopus (329) Google Scholar). DSGs are synthesized as preproproteins, which are processed first in the endoplasmic reticulum to remove the signal sequence and subsequently by the Golgi proprotein convertases to remove the propeptide before transport to the cell surface. The cadherin propeptide is thought to modulate the conformation of the extracellular domains to prevent intracellular aggregation because of interaction with other cadherins within the secretory pathway. Propeptide cleavage occurs upstream of the conserved tryptophan residue at position 2, which is responsible for cadherin strand dimer formation, suggesting that propeptide removal may unmask residues important in intermolecular adhesion. The proprotein convertase furin processes recombinant DSGs in baculoviral overexpression systems (Posthaus et al., 2003Posthaus H. Dubois C.M. Muller E. Novel insights into cadherin processing by subtilisin-like convertases.FEBS Lett. 2003; 536: 203-208Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), which are widely used for pemphigus research and clinical diagnostic purposes. Commercial DSG ELISA kits use baculovirally produced recombinant DSG antigen (Ag) and have been shown to be a sensitive and specific diagnostic tool for pemphigus (Ishii et al., 1997Ishii K. Amagai M. Hall R.P. Hashimoto T. Takayanagi A. Gamou S. et al.Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins.J Immunol. 1997; 159: 2010-2017PubMed Google Scholar). Earlier, pathogenic anti-DSG3 MAbs were isolated from human patients and PV model mice (Amagai et al., 2000Amagai M. Tsunoda K. Suzuki H. Nishifuji K. Koyasu S. Nishikawa T. Use of autoantigen-knockout mice in developing an active autoimmune disease model for pemphigus.J Clin Invest. 2000; 105: 625-631Crossref PubMed Scopus (216) Google Scholar; Payne et al., 2005Payne A.S. Ishii K. Kacir S. Lin C. Li H. Hanakawa Y. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (170) Google Scholar). We have recently observed decreased ELISA binding by some pathogenic PV MAbs, despite consistent pathogenicity against endogenously expressed DSG3 in human keratinocytes. We hypothesized that the variability in pathogenic PV MAb ELISA was because of differential binding of mature DSG3 versus DSG3 proprotein, as the proprotein is commonly observed in recombinant Ags purified from baculoviral overexpression systems. We requested the Ag data from Medical and Biological Laboratories Co., Ltd (MBL International, Woburn, MA), the commercial distributor for DSG ELISA. Interestingly, an increase in purified DSG3 proprotein was observed when Ag production methods switched from stationary plate culture (Figure 1a, lot 012) to roller bottle culture (Figure 1a, lots 013 and 014). Baculoviral roller bottle or spinner culture often results in a higher yield of recombinant proteins than stationary plate systems. However, as suggested by Figure 1a, increased cell lysis associated with these cultures can cause the release of immature DSG3 proprotein into culture supernatants. We evaluated a panel of pathogenic and non-pathogenic human and mouse anti-DSG MAbs (summarized in Figure 1b) for their ability to immunoprecipitate proprotein and mature DSG3 isoforms from recombinant baculoviral culture supernatants. Human pathogenic PV MAbs, P1 and P3, and mouse pathogenic MAb, AK23, selectively immunoprecipitate mature DSG3 (Figure 1c). In contrast, human non-pathogenic MAbs, NP1 and NP2, mouse non-pathogenic MAbs, AK15 and AK18, and one human pathogenic MAb, P2 (which recognizes a non-conformational epitope), immunoprecipitate both mature and proprotein isoforms. To confirm that furin proprotein convertase cleaves the DSG3 propeptide, recombinant DSG3 was purified from baculoviral supernatants by metal affinity chromatography and incubated with furin (20U/mg) for 16hours at room temperature in the manufacturer's recommended buffer (New England BioLabs, Ipswich, MA). Figure 2a shows that furin effectively processes DSG3 proprotein into the mature DSG3 isoform. To evaluate whether altered ratios of DSG3 isoforms affect ELISA binding by anti-DSG3 MAbs, we treated current commercial DSG3 ELISA wells with furin enzyme (2U/well in Tris-buffered saline plus 1mM CaCl2 for 1hour at room temperature) before incubation with anti-DSG3 MAbs. Furin treatment increases the ELISA binding of all human pathogenic MAbs (P1, P2, and P3), as well as the mouse pathogenic MAb, AK23. Furin treatment also modestly increases the binding of human non-pathogenic NP2 MAb, which recognizes a non-conformational epitope in the amino-terminal domain of DSG3 (Figure 2b). Furin treatment shows no significant effect on the binding of other non-pathogenic human and mouse MAbs. As increases in proprotein Ag levels seem to disproportionately decrease the binding of pathogenic versus non-pathogenic PV MAbs, we sought to determine whether the clinical performance of the DSG3 ELISA would be affected by the variability of Ag isoforms. MBL produced custom mature DSG3 ELISA plates by furin treatment of DSG3 before Ag adsorption (as shown in Figure 2a). A pilot study of 85 independent PV patient sera indicates that use of the mature DSG3 Ag does not change the diagnostic result compared with the current DSG3 ELISA. However, in 30 of the 85 samples, use of mature DSG3 ELISA increases the serum index value by 15% or more compared with the current DSG3 ELISA (range 15–33%), whereas only 1 of 85 samples shows a decrease in the index value of ≥15% (value=15%) (demarcated by the 45 degree dashed line in Figure 2c). The mean serum index value increased from 116 to 129 with use of the mature DSG3 ELISA, which was statistically significant by the paired t-test analysis (P=1 × 10−14). Similar binding of DSG3 isoforms between the two kits was confirmed by anti-E tag ELISA (unpublished data). In summary, our results indicate that pathogenic PV MAbs preferentially bind epitopes in mature DSG3 that are masked in the proprotein isoform. In contrast, non-pathogenic anti-DSG3 MAbs recognize both mature and proprotein isoforms, correlating with the binding of non-conformational DSG epitopes. Earlier studies have shown that pathogenic pemphigus antibodies more often bind conformational epitopes in the amino-terminal domain of DSGs, whereas non-pathogenic antibodies bind non-conformational epitopes (Sekiguchi et al., 2001Sekiguchi M. Futei Y. Fujii Y. Iwasaki T. Nishikawa T. Amagai M. Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins.J Immunol. 2001; 167: 5439-5448Crossref PubMed Scopus (143) Google Scholar; Li et al., 2003Li N. Aoki V. Hans-Filho G. Rivitti E.A. Diaz L.A. The role of intramolecular epitope spreading in the pathogenesis of endemic pemphigus foliaceus (fogo selvagem).J Exp Med. 2003; 197: 1501-1510Crossref PubMed Scopus (144) Google Scholar; Payne et al., 2005Payne A.S. Ishii K. Kacir S. Lin C. Li H. Hanakawa Y. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (170) Google Scholar; Ishii et al., 2008Ishii K. Lin C.Y. Siegel D.L. Stanley J.R. Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies.J Invest Dermatol. 2008; 128: 939-948Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Therefore, a predominance of proprotein in the DSG3 ELISA might bias the test to the detection of non-pathogenic antibodies. Although the clinical diagnostic value of the ELISA is unaffected by variability in the DSG3 isoform (Figure 2c), we would predict that the mature DSG3 ELISA would correlate better with disease activity. Our study does not directly evaluate this hypothesis, although a concurrent study supports this conclusion (Yokouchi et al., 2008). Commercial DSG ELISA plates will use mature DSG3 Ag, cleaved with furin before adsorption, beginning in December 2008 (lots 101 and up). Ongoing research studies may note changes in optical density values using the new ELISA kits. These findings are relevant for physicians and scientists using baculovirally produced recombinant DSG3 for clinical and basic research studies, including the use of ELISA to track disease activity and for the evaluation of human and mouse anti-DSG MAbs. Keiko Kuroda and Takahisa Hachiya are employees of the Medical and Biological Laboratories Co., Ltd, the commercial distributor of the desmoglein ELISA. Preety M. Sharma, Eun Jung Choi, Ken Ishii, and Aimee S. Payne declare no conflict of interest. We thank John Stanley, Masa Amagai, and Shinsuke Tachi for helpful discussions on the manuscript. This work was supported by NIH AR053505 and the Jeannette Laws and Thomas McCabe Pilot Award (ASP).